Tick-borne bacterial pathogens of humans cause significant morbidity and mortality throughout the United States and abroad. Lyme disease (LD), caused by Borrelia burgdorferi, is the most prevalent arthropod-borne disease of humans in the United States and many other countries throughout Europe and Asia. Tick-borne relapsing fever (TBRF), caused by Borrelia hermsii, is endemic in scattered foci throughout many regions of higher elevation in the western United States. Our work has focused on four areas: 1) to improve on the serodiagnosis of LD and TBRF fever by using recombinant DNA technology to clone genes of spirochetes that express proteins that induce specific and detectable antibody responses; 2) to examine how spirochetes adapt to their tick and mammalian hosts; 3) perform genetic analysis of spirochete isolates and comparative genomics between relapsing fever and Lyme disease spirochetes; 4) determine the geographic distribution of relapsing fever spirochetes in North America and foci for human infections This work requires that we maintain colonies of Ixodes scapularis and Ornithodoros hermsi, the respective tick vectors of LD and TBRF spirochetes, and infect these ticks via a laboratory mouse - tick cycle. Our interests in serodiagnosis and adaptations associated with tick infection and transmission demand that we have a solid understanding of genetic and phenotypic variation within our species of interest. Genome sequencing projects of the relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed the presence of six open reading frames (ORFs) involved in purine metabolism and salvage that are absent in the Lyme disease spirochete Borrelia burgdorferi. The unique relapsing fever-specific ORFs included genes for hypoxanthine-guanine phosphoribosyl transferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), ribonucleotide reductase alpha subunit (nrdE), and the ribonucleotide reductase beta subunit (nrdF). Southern blot assays confirmed that these genes were restricted to the relapsing fever group of spirochetes. Taqman real-time RT-PCR demonstrated that the chromosomal genes (hpt, purA, purB) were transcribed in vitro and in mice. Phosphoribosyl transferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did B. burgdorferi. B. hermsii exhibited phosphoribosyl transferase activity with adenine, hypoxanthine and guanine. Surprisingly, B. burgdorferi showed low but significant phosphoribosyl transferase activity with hypoxanthine even though the genome lacks a discernible hpt gene. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA, and did so to a much greater extent than did B. burgdorferi. This additional pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to the spirochete's ability to achieve high cell densities in blood. This novel pathway may also lend itself to new therapies for the treatment of relapsing fever. This year we also completed our genetic analysis of the North American species of relapsing fever spirochetes. Isolates of Borrelia turicatae, Borrelia parkeri and the Florida canine borrelia (FCB) were examined to further phylogenetically characterize the identity of these spirochetes in the United States. DNA sequences of four chromosomal loci (16S rRNA, flaB, gyrB, and glpQ) were determined for eight isolates of B. turicatae and six isolates of B. parkeri, which grouped the spirochetes into two distinct but closely related taxa (>98% sequence identity) separate from Borrelia hermsii. Analysis of plasmids demonstrated both linear and circular forms in B. turicatae but only linear plasmids in B. parkeri, which should be of interest to investigators concerned with plasmid diversity and evolution within this group of spirochetes. The Florida canine borrelia clearly separated with the group identified as B. turicatae, confirming this bacterium as a relapsing fever spirochete. Therefore, the potential for tick-borne relapsing fever in humans and other animals exists in Florida, and future efforts are needed to determine the enzootic hosts and distribution of this spirochete in the southeastern United States. We also completed our analysis of 31 isolates of Borrelia hermsii from throughout western North America. The same four chromosomal loci were examined as above, which segregated the species into two distinct clonal lineages, referred to as Genomic Group I and Genomic Group II. The sequence of the gene encoding the variable tick protein (Vtp) was also determine for all isolates, which showed 7 distinct groups. Comparisons of the phylograms demonstrated examples of horizontal transfer of vtp between the two genomic groups. These well-characterized isolates will allow us to now examine superinfections of multiple isolates and strains in mammals and ticks.